研究在CJN实验室由NSERC，加拿大癌症协会研究所（CCSRI），以及加拿大乳腺癌基金会（BC-育空分公司）工作的资助。

注意:这个程序的一般工作流程是在图2列出。 1.测定生长抑制的剂量<ol><li>酵母过夜培养的制备 注:在本协议中所使用的酵母提取物，蛋白胨-葡萄糖培养基（YEPD）是一个标准的配方13。 <ol><li>条纹出BY4741（ 的MATa HIS3Δ1leu2Δ0met15Δ0ura3Δ0）细胞在YEPD琼脂平板孵育48小时，在30℃，或直至可见菌落形成。 </li><li>接种5毫升YEPD液体介质中的无菌培养容器与单个菌落制备过夜培养物。隔夜孵育在30℃，连续旋转或晃动。培养通常由早上（〜2×10 8个细胞/ ml）达到饱和。 </li></ol></li><li>固体琼脂培养基制备含有不同的化学剂量<ol><li>准备几个股票化学将在100倍所需的终浓度在适当溶剂中进行测试。 注:有效浓度将取决于化学，并且必须凭经验确定。因此，建议最初测试了广阔的终浓度范围内， 即从低μM到高的MM。 </li><li>对于化学品的每种浓度进行测试，等分3毫升熔融YEPD琼脂到几个无菌培养管中。发生在55℃水浴，以防止凝固。 </li><li>对于化学的各个浓度待测试添加30微升的100倍的化合物的熔融媒体，涡旋2-3秒，以混合，并且吸管1毫升成每对重复试验孔在12孔板中。一定要包括车辆只能控制。允许板过夜设定为室温。放弃任何额外介质。 </li></ol></li><li>传播电镀细胞<ol><li>接种10毫升YEPD液体介质与2微升饱和酵母培养物，一夜之间成长为在步骤1中。1.2。 </li><li>板25微升稀释酵母培养物每孔，均匀涂抹用无菌玻璃珠或杆，并允许板下火焰干燥。孵育板在30℃下48小时。 </li><li>评价酵母在平板上生长（菌落两者总数和大小）。选择的化合物的亚致死浓度不通过大于10％的抑制生长15％来执行屏幕。 </li></ol></li></ol> 2.系统化学遗传屏幕<ol><li>缺失突变体阵列的维护源板（DMA）和制备<ol><li>有关从甘油股票构建缺失突变体阵列的详细说明请参考Baryshnikova 等 14。一旦缺失突变体已被排列，以每板384菌落的密度时，DMA可以在4℃保存数月。复制在含有作为必要的G418的200微克/毫升，以防止从菌落生长到每个YEPD琼脂等。 注意:多个串行复制应该避免，以防止生长缓慢的品系的损失最小化和抑制的突变体的外观。这一点尤其重要，如果保持藏品的单倍体。 </li><li>执行使用微生物排列机器人系统中的所有副本电镀步骤。另外，操纵阵列使用手动钉扎工具殖民地。 注:酵母删除集合可从几个商业渠道单倍体和二倍体和通常运作为甘油储在96孔板中。 </li></ol></li><li>冷凝的缺失突变体阵列<ol><li>制备250毫升含有G418的200微克/毫升YEPD琼脂介质。 G418的有效浓度可以变化，并且每批应凭经验测试。 </li><li>通过倾倒50毫升含有每板的G418的200微克/毫升YEPD琼脂制备5 YEPD琼脂平板上。冷凝前阵做到这一天，让板冷却，在室温erature隔夜放在平坦的表面上。 注:四板将需要采集在1536株密度，第五板倾作为额外。 </li><li>除去含有以每板384菌落密度的突变体缺失集合16的培养皿，并允许达到室温（约1小时）。 </li><li>擦拭用一个棘手的任务擦拭已形成于各板的盖子的任何冷凝。如果不这样做，可能会导致水滴沉积的阵列和阵列突变体的交叉污染上。 </li><li>使用微生物阵牵制机器人，从384菌落每板（16培养皿）至1536菌落每盘（4培养皿）的密度凝聚DMA。执行此以便从板1引脚的1次菌落到行冷凝的阵列的第1列1，板2的1次菌落到行1列2，板3的第1次菌落到行2和列1，和板4的第一届殖民地行2第2列。 </li><li>孵育统一阵列在30℃下过夜。 </li><li>检查阵列，以确保殖民地从源盘均匀传输。会有若干空白位置，故意掺入到所述阵列，其中作为指导，以确保在DMA已经凝结正确（ 图4A）。 注意:这些将是复制品镀源板​​在含有试验化合物的介质。单个源板可用于多种pinnings。也有不少机器人将有一个对冲的功能，这将确保酵母正在每次传输类似的数字。 </li></ol></li><li>副本板缺失突变体阵列<ol><li>准备700毫升管制（车辆专用）和700毫升实验媒体（含化学品）的。这是足够用于进行一式三份的测定（每个条件12板，加上两个额外）。 </li><li>倒入50毫升YEPD琼脂含测试化学或每盘的控制。允许板吨Ø在室温下冷却过夜平坦的表面上。 </li><li>使用机器人复制品板，接种在DMA上三套含有化学品的试验确定浓度的板，以及三组含的车辆控制的板。孵育板在室温下搅拌24-48小时。 </li></ol></li></ol> 3.成像板和数据分析<ol><li>从孵化器中删除板，并允许他们来室温（约1小时）之前的成像。这将防止凝结而成像板形成。 </li><li>在24和48小时通过移除盖和放置面朝下放在平板扫描仪图像板。在至少300 dpi的分辨率捕捉图像。另外，使用数码相机拍摄的图像。如果这样做，地方板块面朝上放在一个黑暗的背景，取下盖子图像。 注:板的文件格式和定位将取决于所使用的量化方案。 </li&gt; <li>进行量化，用几个开源项目，包括Balony 15，SGAtools 16和ScreenMill 17的一个阵列殖民地大小的比较。 </li></ol> 4.验证屏注:在这个阶段，几个酵母突变体的缺失将比分为过敏感兴趣的化学品。这些化学遗传相互作用必须接下来在两个方面进行验证。第一敏感菌株的身份应通过PCR来确认。二，化学敏感度必须独立评分。下面概述是一个简要的协议用于进行斑点试验，以验证应变超敏反应，在酵母中的生物学共同的技术。 <ol><li>条纹出所需的（过敏性）从酵母缺失突变体集合到含有G418的200微克/毫升YEPD琼脂上。孵育在30℃，直至菌落形成。通过诊断性PCR验证菌株的基因型与根据引制造商的协议。 </li><li>接种将5ml YEPD液体每个菌株的孵育过夜，在30℃下用旋转或摇动。 </li><li>测量OD 600和冲淡每个菌株以OD 600 = 1在无菌的dh 2 O.这些是目前的标准化的酵母培养物。 </li><li>分配100μl的归一化的野生型酵母（BY4741）培养至A1孔96孔板。分配100μl的附加多达七个品系到孔B1-H1。 </li><li>使用多通道移液器通过A6-H6免除90微升无菌的dh 2 O的水井A2-H2。最后，准备了一系列1:10稀释归酵母培养的。开始由串联吹打10微升塔1至第2列与多通道移液器，并继续在96孔板直到6稀释液制成（ 即 10 0到10 -5）。 </li><li>使用多通道传输2-5微升稀释的酵母培养物中的网格移液器到含有化学和车辆控制YEPD固体培养基。孵育板在适当温度下24-48小时。 </li><li>检查板和比较选择的突变体，以化学（相对于BY4741控制）的灵敏度。在24和图像板48小时如在步骤3.2。 注意:虽然几种过敏反应菌株与相关基因本体或功能的识别借给信心到屏幕的有效性，一种过敏反应缺失菌株用含有该基因的野生型拷贝的质粒的转化是必需的正式建立一个基因删除的利息（而不是隐藏第二位点突变）导致药物的敏感性。 </li></ol>

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The authors declare they have no competing financial interests.

这里列出的是产生化学遗传相互作用的配置文件的方法。该方法是简单的:通过比较在对化学品的存在和不存在完整基因缺失集合各菌株的菌落的大小，要容忍化学损伤所需的所有基因被识别。所得敏感菌株的列表的注释富集分析然后可以用于提供洞察行动的化学物质的模式。虽然该协议已被优化的芽殖酵母，它也可以适用于与其他阵列微生物缺失的集合，如大肠杆菌使用大肠杆菌庆...

的遗传工具，并在模式生物酿酒酵母可利用的资源已启用大规模功能基因组学的研究，它们共同提供了新的见解的基因如何充当网络来履行生物系统的要求。这些工具的基础是在酵母1,2-一套完整的所有开放读框的非必需基因缺失的协作创作。一个显着的观察是，只有约20％的酵母基因时，作为标准的实验室条件下培养单倍体所需的可行性。这凸显了一个细胞通过替代生物途径的利用率，减缓基因组扰动的能力。遗传突变体是可行的单独的，但致命的组合，信号连接或收敛平行生物途径和形式描述的生物学功能的基因相互作用网络。带有条件TE的发展必需基因的mperature敏感和亚效等位基因的等位基因的技术还没有被限制在非必需基因3,4的研究。这个概念已经在基因组规模已经应用产生偏见的遗传相互作用地图说明如何参与类似的细胞过程的基因聚集在一起5。 基因网络的化学扰动模拟基因缺失（ 图1）6。查询生长抑制化合物对缺失株的高密度阵列，用于超敏反应标识化学遗传相互作用信息， 即所需要容忍化学应力的基因的列表。像遗传相互作用，化学文库的大规模的屏幕显示，与动作一起簇7的类似模式的化合物。因此，通过建立一个化合物的化学遗传相互作用轮廓的作用方式，可以通过将其与LAR比较推断GE规模合成的遗传和化学遗传相互作用数据集8,9。 大型化学遗传筛选，其中化合物的分数审问，已经由条码竞争测定进行。在这种方法中，缺失菌株的汇总收集于小体积的含有化学介质中生长集体几代。由于每个缺失突变窝藏一个独特的遗传条形码，个别突变体缺失菌株的池内的生存能力/生长被微阵列或高通量测序10跟踪。 通过监测生长在含有生物活性化合物的固体琼脂物理阵列突变体菌落大小推断健身也识别化学遗传相互作用11,12的有效方法。这种方法提供了一种具有成本效益的替代竞争为基础的筛选，非常适合用于测定化学品的小库。这里所概述是用于制造在酿酒化学遗传相互作用的列表的简单方法酵母不依赖于分子生物学操作或基础设施，它仅需要一个酵母删除集合，机器人或手动钉扎装置中，并自由可用的图像分析软件。

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Name of Material/ Equipment&#160;</td>
			<td style="width:161px;">Company&#160;</td>
			<td style="width:156px;">Catalog Number&#160;</td>
			<td style="width:457px;">Comments/Description&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Yeast Extract</td>
			<td style="width:161px;">BioBasic</td>
			<td>G0961</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 1% final concentration (w/v)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:213px;">Tyrptone Powder</td>
			<td style="width:161px;">BD Biosciences</td>
			<td>211820</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">Dextrose</td>
			<td style="width:161px;">Anachemia</td>
			<td style="width:156px;">31096-380</td>
			<td style="width:457px;">For YEPD liquid/solid media add to 2% final concentration (w/v) - Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Agar A</td>
			<td style="width:161px;">Bio Basic</td>
			<td>FB0010</td>
			<td style="width:457px;">For YEPD solid media add to 2% final concentration (w/v)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:213px;">G418</td>
			<td style="width:161px;">A.G. Scientific Inc.</td>
			<td style="width:156px;">G-1033</td>
			<td style="width:457px;">Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">12 well plate</td>
			<td>Greiner Bio One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">5ml culture tubes</td>
			<td>Evergreen Scientific</td>
			<td>222-2376-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">10cm Petri Dish</td>
			<td style="width:161px;">VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROTOR HDA</td>
			<td>Singer Instruments&#160;</td>
			<td>ROT-001</td>
			<td>high-throughput microbial array pinning robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PLUSPLATE&#169; Petri Dish</td>
			<td>Singer Instruments&#160;</td>
			<td>PLU-001</td>
			<td>Box of 200 dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">384 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-384</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1536 Short-Pin RePad</td>
			<td>Singer Instruments&#160;</td>
			<td>RP-MP-1536</td>
			<td>Box of 1000 pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alternative Pinning Tools:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fully Automated Robtic Systems</td>
			<td>S&#38;P robotics</td>
			<td>http://www.sprobotics.com</td>
			<td>Several automated colony handling robitic and imagining systems available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Pinning Tools</td>
			<td>V&#38;P Scientific</td>
			<td>http://www.vp-scientific.com</td>
			<td>Handheld replication tools and accessories.&#160;</td>
		</tr>
	</tbody>
</table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

确定生物活性的化学物质的作用方式是感兴趣的范围广泛的学术，医药，和工业的科学家。 酿酒酵母，或出芽酵母，是一个模型真核生物的量的〜6000基因缺失突变体和亚效等位基因必需基因的完整集合突变体是市售的。突变体的这些集合可以被用来系统地检测化学基因的相互作用，必要即基因容忍的化学物质。这个信息，反过来，报告了化合物的作用可能模式。在这里，我们描述了一个协议，用于快速识别在芽殖酵母化学基因的相互作用。我们证明使用化疗剂5-氟尿嘧啶（5-FU），其具有定义良好的作用机制的方法。我们的研究结果表明，都需要在5-FU的存在下，这是与以往的米一致增殖外来体和DNA修复酶的核TRAMP核糖核酸icroarray基于条形码化学遗传方法和5-FU的RNA和DNA代谢产生不利影响的认识。这些高通量筛选所需的验证方案也有所说明。

作为这种方法的一个验证我们进行化疗剂5-氟尿嘧啶（5-FU），按照以上概述的协议的一个代表性化学遗传相互作用屏幕。 5-FU是已知破坏胸苷酸合成酶，以及DNA和RNA代谢18。 5-FU的化学遗传相互作用是很好的研究，并已研究了通过使用两个杂合和纯合缺失集合8,19酵母条形码微阵列技术。在这里，我们表明，类似的结果可以通过突变菌落大小比较定量来获得。 应...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K 次观看.  University of Victoria. Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

视频: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

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